1. Field of the Invention
The present invention is generally related to the treatment of human beings with pharmacological quantities of hormones and, more particularly, to the use of dehydroepiandrosterone (DHEA) as an inhibitor of platelet function.
2. Description of the Prior Art
Platelets are the blood cells which are instrumental in stopping bleeding when the integrity of a blood vessel is compromised. Normal blood vessels are lined with a single layer of cells called the endothelium. The endothelium regulates vascular tone and prevents platelets from sticking to blood vessel walls. When the endothelial barrier is disrupted, platelets adhere to sub-endothelial components where they become "activated" and elaborate a number of biologically active compounds. One compound produced after the platelets are "activated" is thromboxane A.sub.2 which serves the function of recruiting other platelets to aggregate and form a tight plug called a thrombus. When a vessel has been lacerated, the process of platelet aggregation and thrombus formation serves the beneficial function of stopping the bleeding and allowing time for repair However, if a thrombus forms in the lumen of an intact vessel, the blood flow to tissues subserved by the thrombus becomes compromised and cell death results.
Vascular injury and, in particular, endothelial damage can be caused by many mechanisms. For example, disease processes such as diabetes, hypertension, hyperlipidemia, oxidant injury, and cigarette smoking are known to cause endothelial damage. In addition, endothelial damage can occur iatrogenically (by the actions of a physician) through the use of intravascular remodeling devices such as by balloon angioplasty, atherectomy, or laser surgery. Platelets adhere to areas where the endothelial barrier has been disrupted, and even if an occlusive thrombus does not result when platelets aggregate at the disruption site, the platelets still elaborate compounds thought to be directed toward the repair process such as growth and chemotactic factors which are themselves thought to be important in atherogenesis. Hence, platelet function is thought to play an important role in the generation of atherosclerotic plaques and the events leading to restenosis following mechanical remodeling. It is widely thought that ruptured atherosclerotic plaque is the substrate upon which an acute occlusive thrombus is formed. Intra-coronary thrombosis is the primary etiology of myocardial infarction and stroke.
Platelet function includes a wide variety of platelet activities including among others aggregation, adhesion, and thromboxane production. At present there is a need to find agents capable of attenuating certain platelet activities. Inhibition of platelets could reduce the risk of atherosclerosis, heart attack, stroke, and restenosis following intravascular remodeling procedures. Preferably, suitable agents would not disrupt the ability to stop bleeding in the event of an injury and would not have significant side effects, but would be able to attenuate the deleterious effects of platelets in the disease processes noted above.
While platelets can be activated by a number of biological compounds, many times activation involves a common final pathway which includes the generation of thromboxane A.sub.2. As pointed out above, thromboxane A.sub.2 is instrumental in platelet aggregation and thrombus formation. Thromboxane A.sub.2 is one of the most potent platelet activators known. In addition, thromboxane A.sub.2 is a potent vasoconstrictor. Thus, one mechanism for inhibiting platelet aggregation is to block thromboxane formation. Aspirin is the most widely used drug for inhibiting thromboxane formation and preventing subsequent platelet aggregation. In fact, in large clinical studies aspirin has been shown to reduce the incidence of myocardial infarction. However, aspirin does not inhibit initial platelet adhesion or affect elaboration of compounds such as growth factors which are involved in restenosis. Moreover, aspirin does not appear to have an affect on the platelet's role in atherogenesis.
DHEA is an endogenous androgenic steroid which has been shown to have a myriad of biological activities. In U.S. Pat. No. 4,920,115 to Nestler et al., DHEA was shown to reduce body fat mass, increase muscle mass, lower LDL cholesterol levels without affecting HDL cholesterol levels, lower serum apilipoprotein B levels, and not affect tissue sensitivity to insulin in human patients. In Geriatrics 37:157 (1982), DHEA was reported to be a "miracle drug" which may prevent obesity, aging, diabetes mellitus and heart disease. DHEA was widely prescribed as a drug treatment for many years; however, the Food and Drug Administration recently restricted its use. DHEA is readily interconvertible with its sulfate ester DHEA-S through the action of intracellular sulfatases.
Peak serum DHEA and DHEA-S levels occur when a patient is approximately twenty five years old and decline over the ensuing decades. In Ohrentreich et al., J. Clin. Endocrinol. Metab. 59:551-555 (1984), the mean DHEA-S levels and ranges for adult men and women were characterized. For example, in men the means were reported to be as follows: age 25-29=3320 ng/ml, age 45-49=1910 ng/ml, and at age 65-69=830 ng/ml. Similar age related declines in serum DHEA-S levels were found to occur in women. Correspondingly, the incidence of cardiovascular disease in human beings increases with age, thus suggesting an epidemiological relationship between serum DHEA and DHEA-S levels and cardiovascular disease. In Barrett-Conner et al., N. Engl. J. Med. 315:1519-1524 (1986), the baseline DHEA-S levels of 242 middle aged men (ages ranging between 50 and 79 years) was compared to the subsequent 12 year mortality rate of the men from any cause, from cardiovascular disease, and from ischemic heart disease. DHEA-S levels were significantly lower in men with a history of heart disease compared to those without. In men with no history of heart disease, the age-adjusted relative risk associated with DHEA-S levels below 140 .mu.g/dL was 1.5 (p NS) for death from any cause, 3.3 (p&lt; 0.05) for death from cardiovascular disease, and 3.2 (p&lt;0.05) for death from ischemic heart disease. An increase in DHEA-S level of 100 .mu.g/dL had a 48% reduction in mortality (adjusted for other risk factors) from cardiovascular disease (p &lt;0.05).
Recently, some studies have suggested that DHEA may have anti-atherosclerotic activity and perhaps acts as an anti-proliferative agent. In particular, Arad et al. in Arteriosclerosis 9:159-166 (1989) and Gordon et al. in J. Clin. Invest. 82:712-719 (1988) both describe the reduction of atherosclerotic plaque formation by DHEA. In Arad et al., rabbits were fed 0.5% cholesterol and 0.5% DHEA. The extent to the aortic surface covered by fatty streaks was evaluated after two months. Arad et al. observed a 30 or 40% reduction in fatty streak formation as determined by chemical analysis or planimetry of the lesions, respectively. In Gordon et al., rabbits were fed a 2% cholesterol diet and had arterial injury induced by balloon abrasion. One group was fed 0.5% DHEA, and that group had a 50% reduction in plaque size compared with the rabbits not receiving DHEA. The individual reduction of plaque size was inversely proportional to the serum level of DHEA attained, and was not attributable to differences in body weight gain, food intake, total plasma cholesterol or distribution of cholesterol among the VLDL, LDL, or HDL fractions. Neither Arad et al. nor Gordon et al. made any assessment of the platelet function in their studies and did not hypothesize that alteration in platelet reactivity might have contributed to their findings. Moreover, the data in Arad et al, and Gordon et al. would not support drawing such a conclusion.
In Marks et al., Proc. Natl. Acad. Sci. 46:447-452 (1960), it was reported that DHEA was a specific inhibitor of the enzyme glucose-6-phosphate dehydrogenase which is a proximal enzyme in the hexose monophosphate shunt pathway (HMPS). The HMPS pathway is crucial for generating reducing equivalents, such as the reduced form of .beta.-Nicotinamide Adenine Dinucleotide Phoshphate (NADPH), which are vital to many cellular functions. Marks et al. commented that, with the resulting decrease in NADPH production, inhibiting glucose-6-phosphate dehydrogenase could be a means for regulating steroid and cholesterol biosynthesis.
Another consequence of decreased NADPH production by inhibition of glucose-6-phosphate dehydrogenase is that platelets and other affected cells will have a diminished ability to protect against oxidant damage since they do not have the NADPH required to regenerate reduced glutathione (GSH). Bosia et al. in Thromb. Res. 37:423-434 (1985) examined the consequences of GSH content on platelets using 1-chloro-3,5-dinitrobenzene (CDNB) which chemically depletes GSH resulting in an effect similar to that induced by blocking the enzymatic regeneration of GHS by DHEA. Bosia et al. found that platelets with depleted GSH levels aggregate normally at higher inducer concentrations, have increased or depressed aggregation at low inducer concentrations depending on the inducer, have faster cytoskeletal protein oxidative polymerization but reversible aggregation, do not stimulate the HMPS pathway (the pathway inhibited by DHEA), and are more sensitive to oxidants. Bosia et al. concluded that GSH is a necessary cofactor for platelet thromboxane synthesis and pointed out that similar effects are seen when DHEA is depleted with diamide or in the case of glucose-6-phosphate dehydrogenase deficient cells.
Heffner et al., in J. Clin. Invest., 84:757-764 (1989), disclosed the ability of platelets to attenuate oxidant induced injury in an isolated lung model. Isolated rabbit lungs were perfused with a physiological buffer containing xanthine and xanthine oxidase, a superoxide-peroxide generating system, which resulted in lung damage as measured by the occurrence of lung edema and acute pulmonary hypertension. Inclusion of intact functioning platelets in the perfusate attenuated the injury. Pretreatment of the platelets with DHEA to inhibit glucose-6-phosphate dehydrogenase resulted in augmentation of the lung edema and pulmonary hypertension. Heffner et al. hypothesized that DHEA interfered with antioxidant activity of platelets by blunting the ability to regenerate reduced glutathione. Heffner et al. reported that thromboxane B.sub.2 production was unchanged.
One problem with the Heffner et al. study is that the lungs were perfused with a buffer.+-.platelets solution when, in whole blood, the major oxidant activity resides in the red blood cells and in serum enzymes such as superoxide dismutase. Heffner et al. failed to include such an enzymatic scavenger of superoxide in their controls. The major assessment of lung injury used by Heffner et al. was the measured capillary leak (lung edema). The presence of platelets in the perfusate may attenuate edema by "plugging" capillary leaks. Hence, Heffner et als. hypothesis that DHEA interferes with platelet antioxidant activity by blunting the regeneration of reduced glutathione may be incorrect since DHEA inhibiting the platelet's ability to plug capillary leaks could also explain their results.